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Publikationen - Molekulare Signalverarbeitung

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Publikation

Jablonická, V.; Ziegler, J.; Vatehová, Z.; Lišková, D.; Heilmann, I.; Obložinský, M.; Heilmann, M.; Inhibition of phospholipases influences the metabolism of wound-induced benzylisoquinoline alkaloids in Papaver somniferum L. J. Plant Physiol. 223, 1-8, (2018) DOI: 10.1016/j.jplph.2018.01.007

Benzylisoquinoline alkaloids (BIAs) are important secondary plant metabolites and include medicinally relevant drugs, such as morphine or codeine. As the de novo synthesis of BIA backbones is (still) unfeasible, to date the opium poppy plant Papaver somniferum L. represents the main source of BIAs. The formation of BIAs is induced in poppy plants by stress conditions, such as wounding or salt treatment; however, the details about regulatory processes controlling BIA formation in opium poppy are not well studied. Environmental stresses, such as wounding or salinization, are transduced in plants by phospholipid-based signaling pathways, which involve different classes of phospholipases. Here we investigate whether pharmacological inhibition of phospholipase A2 (PLA2, inhibited by aristolochic acid (AA)) or phospholipase D (PLD; inhibited by 5-fluoro-2-indolyl des-chlorohalopemide (FIPI)) in poppy plants influences wound-induced BIA accumulation and the expression of key biosynthetic genes. We show that inhibition of PLA2 results in increased morphinan biosynthesis concomitant with reduced production of BIAs of the papaverine branch, whereas inhibition of PLD results in increased production of BIAs of the noscapine branch. The data suggest that phospholipid-dependent signaling pathways contribute to the activation of morphine biosynthesis at the expense of the production of other BIAs in poppy plants. A better understanding of the effectors and the principles of regulation of alkaloid biosynthesis might be the basis for the future genetic modification of opium poppy to optimize BIA production.
Publikation

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium, .; Corrigendum: ICTV Virus Taxonomy Profile: Ophioviridae J. Gen. Virol. 99, 949-949, (2018) DOI: 10.1099/jgv.0.001093

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Publikation

Gantner, J.; Ordon, J.; Ilse, T.; Kretschmer, C.; Gruetzner, R.; Löfke, C.; Dagdas, Y.; Bürstenbinder, K.; Marillonnet, S.; Stuttmann, J.; Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system PLOS ONE 13, e0197185, (2018) DOI: 10.1371/journal.pone.0197185

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
Publikation

Stenzel, I.; Ischebeck, T.; Quint, M.; Heilmann, I.; Variable regions of PI4P 5-kinases direct PtdIns(4,5)P2 toward alternative regulatory functions in tobacco pollen tubes Front. Plant Sci. 2, 114, (2012) DOI: 10.3389/fpls.2011.00114

The apical plasma membrane of pollen tubes contains different PI4P 5-kinases that all produce phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] but exert distinct cellular effects. In the present example, overexpression of Arabidopsis AtPIP5K5 or tobacco NtPIP5K6-1 caused growth defects previously attributed to increased pectin secretion. In contrast, overexpression of Arabidopsis AtPIP5K2 caused apical tip swelling implicated in altering actin fine structure in the pollen tube apex. AtPIP5K5, NtPIP5K6-1, and AtPIP5K2 share identical domain structures. Domains required for correct membrane association of the enzymes were identified by systematic deletion of N-terminal domains and subsequent expression of fluorescence-tagged enzyme truncations in tobacco pollen tubes. A variable linker region (Lin) contained in all PI4P 5-kinase isoforms of subfamily B, but not conserved in sequence, was recognized to be necessary for correct subcellular localization of AtPIP5K5, NtPIP5K6-1, and AtPIP5K2. Deletion of N-terminal domains including the Lin domain did not impair catalytic activity of recombinant AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 in vitro; however, the presence of the Lin domain was necessary for in vivo effects on pollen tube growth upon overexpression of truncated enzymes. Overexpression of catalytically inactive variants of AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 did not influence pollen tube growth, indicating that PtdIns(4,5)P2 production rather than structural properties of PI4P 5-kinases was relevant for the manifestation of growth phenotypes. When Lin domains were swapped between NtPIP5K6-1 and AtPIP5K2 and the chimeric enzymes overexpressed in pollen tubes, the chimeras reciprocally gained the capabilities to invoke tip swelling or secretion phenotypes, respectively. The data indicate that the Lin domain directed the enzymes into different regulatory contexts, possibly contributing to channeling of PtdIns(4,5)P2 at the interface of secretion and actin cytoskeleton.
Bücher und Buchkapitel

Vaira, A. M.; Gago-Zachert, S.; Garcia, M. L.; Guerri, J.; Hammond, J.; Milne, R. G.; Moreno, P.; Morikawa, T.; Natsuaki, T.; Navarro, J. A.; Pallas, V.; Torok, V.; Verbeek, M.; Vetten, H. J.; Family - Ophioviridae (King, A. M. Q., et al., eds.). 743-748, (2012) DOI: 10.1016/B978-0-12-384684-6.00060-4

This chapter focuses on Ophioviridae family whose sole member genus is Ophiovirus. The member species of the genus include Citrus psorosis virus (CPsV), Freesia sneak virus(FreSV), Lettuce ring necrosis virus (LRNV), and Mirafiori lettuce big-vein virus (MiLBVV).The single stranded negative/possibly ambisense RNA genome is divided into 3–4 segments, each of which is encapsidated in a single coat protein (43–50 kDa) forming filamentous virions of about 3 nm in diameter, in shape of kinked or probably internally coiled circles of at least two different contour lengths. Ophioviruses can be mechanically transmitted to a limited range of test plants, inducing local lesions and systemic mottle. The natural hosts of CPsV, ranunculus white mottle virus (RWMV), MiLBVV, and LRNV are dicotyledonous plants of widely differing taxonomy. CPsV has a wide geographical distribution in citrus in the Americas, in the Mediterranean and in New Zealand. FreSV has been reported in two species of the family Ranunculacae from Northern Italy, and in lettuce in France and Germany. Tulip mild mottle mosaic virus (TMMMV) has been reported in tulips in Japan. LRNV is closely associated with lettuce ring necrosis disease in The Netherlands, Belgium, and France, and FreSV has been reported in Europe, Africa, North America and New Zealand.
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